SEXUAL HORMONES 2 LEVEL IN PLASMA by LC/MS - Code LC72210
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SEXUAL HORMONES 2° LEVEL IN PLASMA by LC/MS – Code LC72210
(Dihydrotestosterone, Androsterone, Estrone, Testosterone, Estradiol, Androstenedione)
INTRODUCTION
Hormones and hormone action mechanisms
The term "hormone" means a substance that is produced by an endocrine cell, that is, of internal secretion,
is released into the bloodstream, causing functional responses in cells located at various distances from its
production headquarters. For the fulfillment of hormone action are needed, in addition to the synthesis and
secretion, transport into the bloodstream and target in target tissues where the receptors are present,
specialized structures that recognize the specific stimulus and translate the message.
The receptors may be on the cell membrane or within it; the hormone that can not cross the membrane (eg.,
A peptide) binds to receptors located on the plasma membrane, while that which diffuses through the plasma
membrane to the interior of the cell (steroids, iodothyronines) binds to intracellular receptors (typically
located in the nucleus).
Regardless of the structure and the type of hormone receptors have characteristics in common: they all
feature a region able to recognize and bind the hormone and another MEP to the generation of an
intracellular signal that translates the message hormone in functional responses of the target cell; also the
properties that regulate the hormone binding (affinity, specificity, saturability, transduction capacity, ie to
evoke specific effects) are common for all the receptors.
The communication entrusted to hormones takes place for the most part through the bloodstream (endocrine
action), but, to a lesser extent, also by means of other methods. Some hormones act in fact on the cells
immediately surrounding the cell that produces them (paracrine action), in other circumstances, however,
interact with the same cell secretory (autocrine action), other hormones, finally, are produced by the neurons
of the nervous system (action neurocrina , which in reality is a specialized form of paracrine action).
Were identified more than fifty hormones, whose functional characteristics are determined by the different
molecular structure. Based on this, they are divided into four broad categories: proteins and peptides,
steroids, derived from amino acids, derived from polyunsaturated fatty acids. The hormones, interacting with
receptors located at the level of target tissues, evoke specific responses regulate the enzymatic activities,
gene expression and protein synthesis.
Classification steroid hormones
They are fat-soluble, diffuse freely within the cell and exert their action after binding to receptors located in
the nucleus. Their chemical structure, which is polycyclic, derived from cholesterol. They are divided,
according to the site of production, adrenal and gonadal steroids, are included in this category also vitamin D
and its analogues. The steroid hormones produced by the adrenal gland and gonads are divided into
subgroups according to the number of carbon atoms of the steroid nucleus: progesterone, glucocorticoids
and mineralocorticoids derive for subsequent synthesis by pregnane, a simple substance that contains 21
carbon atoms, while estrogen come from the core of estrane which has 18 carbon atoms, and androgens of
androstane from the nucleus, which contains 19 carbon atoms.
The synthesis of steroid hormones following stages biosynthetic identical in both the adrenal and ovary or
testis and differentiation in the three endocrine glands depends on a different distribution of tissue-specific
enzyme synthesis. Steroidogenesis passes through a series of enzymatic steps, mostly catalyzed by
1cytochrome P450 enzymes (cP450) localized within cells, and starts from the conversion of cholesterol to
pregnenolone. This stage enzyme is the most important as it controls the synthesis of all steroid hormones,
even in the presence of significant amounts of cholesterol, the possibility of the continuation of the
biosynthetic pathway appears to be limited, since it depends on the activity by enzymes cP450.
Pregnenolone format comes out of the mitochondria and is transferred to the endoplasmic reticulum, where it
undergoes subsequent enzymatic modifications by cP450. Pregnenolone is then the common precursor of
the major steroid hormones.
Gli androgeni vengono sintetizzati dalle cellule di Leydig del testicolo, dalla zona reticolare del surrene e
dalle cellule della teca del follicolo e dall'interstizio dell'ovaio. La via biosintetica che procede dal
pregnenolone segue due possibili direzioni: la prima via consiste nella trasformazione del pregnenolone in
17-idrossipregnenolone, di questo in deidroepiandrosterone, quindi in androstenediolo, e infine in
testosterone, che è il principale ormone maschile. La seconda via consiste nella trasformazione del
pregnenolone in progesterone, di questo in 17-idrossiprogesterone e quindi nella formazione di
androstenedione, a sua volta successivamente trasformato in testosterone. In alcuni tessuti il testosterone
necessita di un'ulteriore reazione di trasformazione in 5α-diidrotestosterone per esplicare la sua attività. Nel
surrene dove, rispetto al testicolo, vi è una diversa distribuzione degli enzimi che catalizzano queste vie
biosintetiche, la maggior parte della produzione di androgeni surrenali è diretta verso l'elaborazione di
precursori del testosterone e, in maniera particolare, di deidroepiandrosterone e androstenedione, che
hanno un'attività ormonale di più modesta entità. Circa il 50% del pregnenolone metabolizzato nella
corteccia surrenale viene convertito in deidroepiandrosterone.
Gli estrogeni e il progesterone, i principali ormoni femminili, sono gli steroidi prodotti in maggiore quantità
dall'ovaio e sono coinvolti nella regolazione del ciclo mestruale e nella gravidanza. La formazione di questi
steroidi deriva da tappe enzimatiche diversificate nei compartimenti cellulari dell'ovaio: follicoli e cellule
interstiziali. Le cellule della granulosa, che contornano l'ovulo nel contesto del follicolo, subiscono la
trasformazione in cellule luteiniche dopo l'ovulazione e il prodotto della trasformazione del pregnenolone è
rappresentato dal progesterone. Le cellule della teca del follicolo e quelle interstiziali producono in
prevalenza androstenedione attraverso le vie biosintetiche che sono già state descritte per gli androgeni.
Anche le tappe enzimatiche coinvolte nel successivo destino metabolico dell'androstenedione hanno una
caratteristica localizzazione cellulare: infatti il testosterone è sintetizzato solamente nelle cellule dell'ilo,
mentre all'interno delle cellule della granulosa l'androstenedione viene trasformato in testosterone e a sua
volta in estrone e estradiolo. Le cellule interstiziali hanno anche la capacità di trasformare il testosterone in
diidrotestosterone.
2In contrast, peptide hormones, the secretion of steroids in the circulation does not proceed through their
storage within the cells but immediately follows the synthesis. They travel in the plasma bound to specific
transport proteins, with high affinity, such as cortisol-binding globulin (CBG, Cortisol binding protein), the sex
steroid binding globulin (SHBG, Sex hormone binding protein)-binding protein and vitamin D (DBP, vitamin D
binding protein). The CBG is a glycoprotein able to bind with equal affinity cortisol and progesterone. The
SHBG are globulins that bind with high affinity testosterone, whereas estradiol is bound mainly to albumin.
98% of the gonadal steroids, 95% and 50% of cortisol circulating aldosterone linked to the respective
transport proteins. Since only the free hormone is able to interact with the receptors, and then to express the
biological activity, the binding plasma is an important phase of reserves in the metabolism of these
hormones. Synthetic steroids used in therapy usually do not contract bond with transport proteins and are
therefore able to exert biological effects immediate. Glucocorticoids and mineralocorticoids are metabolized
through enzymatic reactions that result in the loss of their hormonal activity or through conjugation with
chemical groups that make them water-soluble and determine its elimination in the urine. The only metabolic
reaction that does not result in a loss of biological activity is represented by the conversion of testosterone to
dihydrotestosterone in target cells.*
*Bibliografy
Enciclopedia della Scienza e della Tecnica (2007)
Andreani, Cassano 1977: Andreani, Domenico - Cassano, Cataldo, Trattato italiano di endocrinologia, Roma, SEU,
1977.
Becker 1995: Becker, Kenneth L., Principles and practice of endocrinology and metabolism, 2. ed., Philadelphia,
Lippincott, 1995 (1. ed.: 1990).
Faglia 1998: Faglia, Giovanni, Malattie del sistema endocrino e del metabolismo, 2. ed., Milano, McGraw-Hill, 1998
(1. ed.: 1993).
Felig 1995: Felig, Philip - Baxter, John D. - Frohman, Lawrence A., Endocrinology and metabolism, 3. ed., New York,
McGraw-Hill, 1995 (1. ed.: 1981).
Giusti, Serio 1988: Giusti, Giorgio - Serio, Mario, Endocrinologia, fisiopatologia e clinica, Firenze, USES, 1988.
Pinchera 1991: Pinchera, Aldo e altri, Endocrinologia e metabolismo. Fisiopatologia e clinica, Milano, Ambrosiana,
1991.
Williams 1998: Williams, Robert H., Williams textbook of endocrinology, 9. ed., edited by Jean D. Wilson e altri,
Philadelphia-London, Saunders, 1998 (1. ed.: 1950).
3Biosynthetic pathway diagram
STEROID HORMONE PROFILES
Steroid hormones have been organized and divided into 4 profiles in order to highlight the four-level adrenal
biosynthesis pathways leading that result in the hormone that determines the final effect at the level of target
tissues.
The primary profiles are the following:
1. Profile Glucocorticoids
2. Profile Mineralocorticoids
3. Profile Sexual Hormones Level 1
4. Profile Sexual Hormones Level 2
In this way it is possible to cover all the needs of endocrinology of the adrenal glands with a detail that would
have been unthinkable a short time ago.
The profile 3 also contains two sex hormones mainly caused by extra-adrenal: Testosterone and Estradiol
that can give a quick information depending on the age and sex of the subject examined.
In a way, act as a litmus test of the central synthesis of sex hormones compared to a dysfunction of
peripheral-type ie gonadal tissue that after what is most responsible for the synthesis of molecules similar.
4This profile can be built from the 2nd level both male and female. The first definitely has a huge importance
for all those events that affect the peripheral utilization of sex hormones, especially androgens in women and
estradiol in the male. Dysfunctions in this level give rise to a whole series of such signs, hirsutism, alopecia,
acne, seborrhea etc.. Such a profile would surely have a great importance for both pediatricians,
gynecologists, urologists, but also dermatologists which would enjoy a tool biochemical able to differentiate
the type of response of the hair bulb compared to the sebaceous gland in the abovementioned events. The
Profile Level 2 female, if carried to adequate levels of sensitivity, could be useful in defining the degree of
conversion of testosterone to estradiol by the adipose tissue and account for example the appearance of
gynecomastia in male patients suffering from obesity as well as monitoring therapy.
EUREKA srl – LAB DIVISION Head Quarter:
VAT N° 01547310423 Via Enrico Fermi 25
E-mail:info@eurekaone.com 60033 Chiaravalle (AN) ITALY
www.eurekaone.com Tel. +39 071 7450790
Fax + 39 071 7496579
This product fulfills all the requirements of Directive 98/79/EC and Dl.ivo 08/09/2000 n.332 on in vitro diagnostic medical
devices (IVD). The declaration of conformity is available upon request.
Release N° 002 Sexual Hormones 2° level in plasma by LC/MS November 2013
5TECHNICAL FEATURES
Principle of Method:
Sexual Hormones are deproteinized with an appropriate reagent containing the internal standard, centrifuged, diluted
and injected in LC/MS.
Recovery : 100%
Sensitivity (LLOD) : Androstenedione 0,001 ng/ml
Androsterone 0,0003 ng/ml
Dihydrotestosterone 0,007 ng/ml
Estradiol 0,001 ng/ml
Estrone 0,0006 ng/ml
Testosterone 0,001 ng/ml
Sensitivity (LLOQ) : Androstenedione 0,003 ng/ml
Androsterone 0,001 ng/ml
Dihydrotestosterone 0,02 ng/ml
Estradiol 0,002 ng/ml
Estrone 0,002 ng/ml
Testosterone 0,003 ng/ml
Dynamic Range : Androstenedione 0,003 - 100 ng/ml
Androsterone 0,001 - 100 ng/ml
Dihydrotestosterone 0,02 - 150 ng/ml
Estradiol 0,002 - 50 ng/ml
Estrone 0,002 - 20 ng/ml
Testosterone 0,003 - 150 ng/ml
ANALYTE Accuracy intra-day Precision intra-day CV% inter-day
(Error %) (CV%) (Error %)
Calibrator Calibrator Calibrator
Low Medium High Low Medium High Low Medium High
Androstenedione 6,3% 4,2% 3,9% 10,2% 5,6% 5,6% 11,6% 5,9% 6,0%
Androsterone 12,9% 4,5% 6,1% 15,4% 5,4% 8,7% 16,7% 5,7% 9,1%
Dihydrotestosterone 10,0% 7,0% 5,2% 13,9% 8,8% 7,3% 13,9% 8,8% 7,5%
Estradiol 11,1% 5,6% 4,8% 12,8% 6,8% 6,1% 14,1% 9,6% 6,9%
Estrone 20,0% 4,5% 4,1% 14,4% 5,8% 4,6% 15,6% 6,7% 5,4%
Testosterone 10,5% 5,3% 4,8% 13,6% 7,2% 5,6% 15,4% 7,4% 5,8%
ANALYTE Concentrations used to calculate precision and accuracy
Calibrator
Low Medium High
Androstenedione 0,1 0,8 10
Androsterone 0,2 1,6 20
Dihydrotestosterone 0,15 1,2 15
Estradiol 0,05 0,4 5
Estrone 0,02 0,16 2
Testosterone 0,15 1,2 15
6Components of the kit (50 tests) : All the reagents are stable 3 years at 2–8 °C.
Reagent A – Deproteinization Solution, 5 x 5 ml
Reagent B – Internal Standard Solution, 1 x 250 µl Store at -20
Reagent C – Diluting Solution, 1 x 3 ml
Reagent D1 – Calibrator in plasma – Level 1, 1 x 1 ml See Warnings
Reagent D2 – Calibrator in plasma – Level 2, 1 x 1 ml See Warnings
Reagent D3 – Calibrator in plasma – Level 3, 1 x 1 ml See Warnings
Reagent D4 – Calibrator in plasma – Level 4, 1 x 1 ml See Warnings
Reagent D5 – Calibrator in plasma – Level 5, 1 x 1 ml See Warnings
Reagent M1 – Mobile Phase M1, 1 x 500 ml
Reagent M2 – Mobile Phase M2, 1 x 500 ml
Minimum Instrumental equipment required: LC/MS System with triple quadrupole medium/high
level (S/N of at least 10.000/1 for 1 pg of injected
Reserpine)
MRM Work Mode, positive APCI
Centrifugal evaporator
Blood Collection Procedure: Take 3 ml of whole blood in a test tube without gel or
with EDTA as anticoagulant. Centrifuge at 4000 rpm
for 5 minutes. Separate the serum and store at – 20
°C. Stable 4 weeks.
7ANALYTICAL PROCEDURE
IMPORTANT: DO NOT USE GLASS TUBES OR VIALS DURING ANY STEP PREPARATION.
STEP 1 : Reconstitution of Deproteinization Solution
• Add at Reagent A – Deproteinization Sol. 50 µl of Reagent B – Internal Std Sol. (Stable 6 months at
- 20 °C)
STEP 2 : Preparation of samples, calibration and control
In eppendorf tubes dispense in sequence:
Calibrator Sample Control
Reagent D1-D5 – 200 µl
Calibrator
Sample 200 µl
Control 200 µl
Reagent A – 500 µl 500 µl 500 µl
Deproteinization Sol (after
reconstitution at STEP 1)
Deproteinize directly on vortex for 20 seconds
STEP 3 : Centrifuge at 14.000 rpm for 10 minutes
STEP 4 : Take 500 µl of surnatant and dry (stable1 month at – 20 °C)
STEP 5 : Add 50 µl of Reagent C – Diluting Solution
Vortex for 1 minute
STEP 6 : Centrifuge at 14.000 rpm for 10 minutes.
Transfer all the supernatant in vials with polypropylene insert
INJECTION :
• Inject 10-20 µl of solution in LC system
N.B: at this step the sample is stable 2 days at 2-8 °C.
Release N° 002 Sexual Hormons 2° level in plasma by LC/MS November 2013
8SEXUAL HORMONES 2° LEVEL - Warnings
REAGENT D1-D2-D3-D4-D5 : CALIBRATOR LYOPHIL. Lot. 004
LEV. 1 LEV. 2 LEV. 3 LEV. 4 LEV. 5
(ng/ml) (ng/ml) (ng/ml) (ng/ml) (ng/ml)
DIHYDROTESTOSTERONE 0,08 0,9 1,2 5,8 13,9
ANDROSTENEDIONE 0,05 0,6 0,8 3,9 7,5
ANDROSTERONE 0,08 1,2 1,4 7,3 13,3
ESTRONE 0,01 0,1 0,14 0,8 1,7
ESTRADIOL 0,03 0,3 0,4 2,2 4,8
TESTOSTERONE 0,05 0,9 1,2 5,9 10,4
Use and Reconstitution: Blood
Calibrators are used for calibration of the
LC system. Add exactly 1 ml of H2O
HPLC grade and mix for 30 min untill all
material is dissolved. Stir gently before
sampling to ensure homogeneity.
Storage and stability: 36 months if
stored at 2 – 8 °C. After reconstituion 7
days if stored at 2 – 8 °C and 6 months
at – 20 °C. Don’t use after expiry date.
Packaging : 5 x 1 ml
Warning: The calibrator derives from
human matrix, so it could be potentially
infected. It must be handled with care.
COLUMN CONDITIONING
Install a new analytical column RRHD Eclipse Plus C18 (50 x 2,1 mm, 1,8 um), termostatate at 50 °C
Disconnect the detector and flux a solution of Methanol : H2O (80 : 20 v/v) at flow of 300 ul / minute for
20 minutes. Don’t recycle the washing solutions. Condition further on the column at the flow of
Mobile Phase M1 : Mobile Phase 2 (50 : 50 v/v) at flow ofi 400 ul / minute for 30 minutes. Two
injections of HPLC grade water before proceeding with the analytical series.
It’s NOT possible to make analysis at recycling phase.
If room temperature is > 20 °C store the Mobile Phase at 2-8 °C between an analytical session and
another.
COLUMN CLEANING
Wash with a solution of Methanol : H2O (80 : 20 v/v) at flow of 300 ul / minute for 10 minutes. Store the
column in a solution of Methanol : H2O (80 : 20 v/v).
INJECTION NEEDLE WASHING
Wash with a solution of Methanol : H2O (80 : 20 v/v).
9FLOW SET
GRADIENT
Time (min) % M1 (PUMP A) % M2 (PUMP B) Flow (µl/min)
0 75 25 600
0.38 75 25 600
5.50 0 100 600
5.90 0 100 600
6.00 75 25 600
7.50 75 25 600
Fragmentations (optimized on AB SCIEX QTRAP® 4500)
COLLISION
ENERGY
Analyte Q1 Q3
Androstenedione 30
273,1 255
Androsterone 20
273,1 255,2
Dihydrotestosterone 20
273,1 255
Estradiol 25
255 159,1
Estrone 26
271,2 133
Testosterone 25
289,1 97
ACCESSORIES AND CONSUMABLES
CODE DESCRIPTION PACKAGING
LC72216 Calibrator in plasma for Sexual Hormones 2° level 5 x 2 x 1 ml
LC72317 Control in plasma for Steroid Hormones – Level 1 5 x 1 ml
LC72318 Control in plasma for Steroid Hormones – Level 2 5 x 1 ml
LC72319 Control in plasma for Steroid Hormones – Levels 1 and 2 2 x 5 x 1 ml
SK72210 Starter kit for Glucocorticoids 1 Pz
Analytical Column RRHD Eclipse Plus C 18 (50 x 2.1 mm. 1.8
Z959757902 1 Pz
um)
S29057U Glass vials of 2 ml with screw cap 1 x 100 Pz
S24722 Polypropylene Insert 100 ul for vials of 2 ml 1 x 100 Pz
10BIBLIOGRAFY:
Quantification of corticosteroids in human plasma by liquid chromatography-thermospray mass
spectrometry using stable isotope dilution
Hiromi Shibasaki, Takashi Furuta, Yasuji Kasuya
Steroid Hormone Analysis by Tandem Mass Spectrometry
Steven J. Soldin and Offie P.Soldin
Quantification of anabolic hormones and their metabolites in bovine serum and urine by liquid
chromatography-tandem mass spectroscophy
R. Draisci, L. Palleschi, E. Ferretti, L.Lucentini, P. Cammarata
Identification of ten corticosteroids in human hair by liquid chromatography-ionspray mass
spectrometry
V. Cirimele, P.Kintz, J.P. Goullè, B. Ludes
A Confirmatory HPLC-MS/MS Method for Ten Synthetic Corticosteroids in Bovine Urines, JOURNAL
OF MASS SPECTROMETRY
S. Rhea Savu, L. Silvestro, A. Haag and F. Sorgel, VOL. 31, 1351-1363 (1996).
Automated solid-phase extraction for concentration and clean-up of female steroid hormones prior
to liquid chromatography–electrospray ionization–tandem mass spectrometry.
B. Alvarez Sanchez, F. Priego Capote, J. Ruiz Jimenez, M.D. Luque de Castro. An approach to lipidomics,
Journal of Chromatography A, 1207 (2008) 46–54.
Detection, quantification and confirmation of anabolic steroids in equine plasma by liquid
chromatography and tandem mass spectrometry
Fuyu Guan, Cornelius E. Uboh, Lawrence R. Soma, Yi Luo, Jeffery Rudy, Thomas Tobin., Journal of
Chromatography B, 829 (2005) 56–68.
Liquid chromatography–electrospray ionization mass spectrometric analysis of corticosterone in rat
plasma using selected ion monitoring,
Ashok Marwah, Padma Marwah, Henry Lardy. Journal of Chromatography B, 757 (2001) 333–342.
Quantification of corticosteroids in human plasma by liquid chromatography–thermospray mass
spectrometry using stable isotope dilution,
Ashok Marwah, Padma Marwah, Henry Lardy, Journal of Chromatography B, 692 (1997) 7–14
11SEXUAL HORMONES 2°LEVEL IN PLASMA
(Reference Chromatograms)
7.2
1.25 7.0
6
1.20
5 6
1.15 6.5
5
1.10
5 6
6.0
1.05
5 6
1.00
5 5.5
9.50
5 6
9.00
4 5.0
8.50
4 6
8.00
4 4.5
7.50
4 6
4.0
7.00
4
6
6.50
4 3.5
6.00
4 6
5.50
4 3.0
5.00
4 6
4.50
4 2.5
4.00
4 6
3.50
4 2.0
3.00
4 6
1.5
2.50
4 6
2.00
4 1.0
1.50
4 6
1.00
4 5.0
5000
4 5
00 0. 0.
0. 1. 1. 2. 2. 3. 3. 4. 4. 5. 5. 6. 0. 1. 1. 2. 2. 3. 3. 4. 4. 5. 5. 6.
00 Time, 0 Time,
5 0 5 0 5 0 5 0 5 0 5 0 5 0 5 0 5 0 5 0 5 0 5 0
i i
Fig. 1 : Calibrator Level 1 Fig. 2 : Calibrator Level 5
R.T. 3.48 ANDROSTENEDIONE R.T. 3.48 ANDROSTENEDIONE
R.T. 3.51 ESTRONE R.T. 3.51 ESTRONE
R.T. 3.62 ESTRADIOL R.T. 3.62 ESTRADIOL
R.T. 3.63 TESTOSTERONE R.T. 3.63 TESTOSTERONE
R.T. 4.1 DIHYDROTESTOSTERONE R.T. 4.1 DIHYDROTESTOSTERONE
R.T. 4.28 ANDROSTERONE R.T. 4.28 ANDROSTERONE
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