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This experiment explains about the anabolic steroids hormones present in human body, their functions and properties. The effects of anabolic steroids are more likely demonstrated on males than in female (reference: john - alder, Cox RM, 2005). Anabolic steroids in male will help in production testis, prostate as well as it will increase the growth in secondary sexual characteristics like muscles, hair etc...,(reference: john - alder, Cox RM, 2005). Some anabolic steroid hormones are very essential for normal sperm production in males and the main steroid used for this is testosterone. Testosterone steroids will also produced in brain equally in both the sexes. The proteins in the human body or in any other living being consists of nitrogen as one component and muscles are developed by proteins, this testosterone will enhance the nitrogen (www.succezz.com/S2/ACompleteGuidtotheUseandAbuseofSteroids). Steroids are injected to animals like cattle in order to improve the meat production by showing the change in growth and feed conversion in cattle, among them boldenone is used most common (reference: http://articles.muscletalk.co.uk/article-boldenone.aspx). Testosterone and boldenone are similar except one difference that is boldenone has double ring every first position of A - ring in the steroid structure (reference: http://articles.muscletalk.co.uk/article-boldenone.aspx). But both are distinguished by their mode of action, effect and metabolism. The structures of various steroids are showed below.
TETSOSTERONE. 2. NANDROLONE.
3. STANOZOLOL. 4. TRENBOL.
5. METHENOLONE. 6. BOLDENONE.
Trebolone is another steroids present in human body. Actually trebolone is derivative of nandrolone. Trebolone consists of two additional double bonds at position 9 and 11 in its steroid structure, Presence of this double bond makes trebolone more binding potency than nadrolone (reference: William liewellyn, 20th February 2006). Actually this trebolone properties are fare differ from its parent hormones (reference: Williams liewellyn, 20th February 2006). Steroids are of two types, one is natural steroids and another one is pharmaceutical steroids. It is generally accepted that chemical testing of biological fluid is the most objective means of diagnosis of drug use (reference: min shen, xiang). Here I have referred various articles on anabolic steroids, which are published recently. From these article i got know that the steroids are also could be detected from animal tissues by doping the drug. There are various types of steroids exist in human body, which can be detected using most sensitive methods (min shen, ping xiang). Scientists developed many methods for this research like liquid chromatography - mass spectrometry, gas chromatography - mass spectrometry, hypertrophy, time of fall etc....,. Here the chromatography is the most widely using method for anabolic steroids detection. Chromatography could be used either ways i.e. for liquid sample and also for gaseous. The different samples present in the extract would have different absorptions characteristics in a solid and liquid phase medium (reference: deng xs, korosu A). The natural steroids are developed within the human body, which is essential to develop energy to do work. Steroids are present in all parts of human being like blood, hair, urine, and in all organs. The steroids are present not only in human beings, but also in all living beings like animals. The steroid determining tests are also conducted on animals also. Anabolic steroids, generally used as therapeutic agents in clinical practice, are also widely abused as performance enhancing drugs. Steroids also manufacture artificially in laboratories known as pharmaceutical steroids, which are used to develop an extra energy and for body development. The consumption of pharmaceutical steroid will be dangerous to health. Extra doping of these artificial steroids may also leads to death. These artificial steroids are also called anabolic steroids. Doping with endogenous steroids became the most serious issue in all sports and competitions. Using of these anabolic steroids are banned by international Olympic committee (IOC) in the year 1975, testing of anabolic steroids is necessary in order to differentiate between pharmaceutical steroids and natural steroids. Why because ,these steroids using wrestlers are getting more energy and power than when they are in normal. Some athletes take anabolic steroids from long time, mostly in winter season and will stop before the competitions, like 4 to 18 weeks before. We cannot detect the drug in take of long time back, but it is necessary find a matrix to detect anabolic steroids. To control the usage of anabolic steroids IOC started testing by keeping the wrestler under observation days before the competitions and performing the standard doping control in laboratories. In endogenous, testosterone, epitestisterone, boldenone, nandrolone, methyltestosterone, methenolone, stanozolol, 6b - hydroxymetandienone, 3 - hydroxystanozolol, DHEA - 2TMS, testosterone - D3 - 2TMS are very important. In forensic department and many occupational and clinical situations hair samples are use to detect the drug intake and many, here also we use the hair in detection of endogenous steroid intake by wrestlers or any others in competitions (reference: J. Segura, S. Pichini). This experiment may take a week or weeks or months which depend up length of the hair that is why hair analysis will be done during training and abstinence. We can also use urine to detect anabolic steroids, but we can fine only few steroids like etiocholanalone, testosterone, androsterone, DHEA, the remaining steroids are invisible in urine and these steroids are detectable within 2 or 3 days of doping. So, hair is well suitable for the experiment, as steroids are traceable long after the intake of anabolic steroids. Doping analysis is not easy all the time there are still some problems using hair analysis, like sometimes anabolic steroids are found in the pictogram per milligram rang and opiates are also some times found in monograms' per milligrams ( min xiang, ping xiang). For these kinds of situations we need more sensitivity method.
The various articles are reviewed and extracted some information regarding the methods, sample preparation and for procedure. Some experiments are also done on animals by doping drug in to them. Doing of anabolic steroid hormones in o animals like cattle will improve the meat production by changing its growth and feeding procedure (reference: john - alder, Cox RM, 2005). For the quantitative determination of eight anabolic steroids in human hair some sensitive, reproducible, specific methods are developed using liquid chromatography / mass spectrometry and gas chromatography/ mass spectrometry. Only some of the steroids are identified by using liquid chromatography/mass spectrograph, like boldenone,nandrolone,methandienone,trenbolone,epitestosterone, methyltestosterone, methenolone, stanozolone. Remaining steroids like DHEA - 2TMS and testosterone - D3 - 2TMS are identified by using gas chromatography/ mass spectrometry.
B. METERIALS AND METHOD:
HUMAN HAIR SAMPLE:
The human hair sample collected from the roots of human head. The hair was cut using round pointed scissors through vertex posterior of the scalp. This hair sample is preserved in closed environment to avoid from the dust and all and stored in room temperature (min shen, ping xiang). The animal hair could also be used for anabolic steroid hormones detection (reference: min shen, ping xiang).
2. CHEMICALS USED:
0.1% of sodium dodecyl sulphate, Acetone, distilled water, 1M NaOH, 1M HCL, sodium phosphate buffer (PH=6.8), diethyl ether, acetonitrile, ammonium acetate buffer, N - methyl - N - trimethyacetamide, iodotrimethylsilane, DL - dithiothreitol.
3. CHEMICAL PROPERTIES AND SAFETY:
Each chemical used in sample preparation has its own concentration, boiling point and even some chemicals are toxic and corrosive. For example, the acetonitrile used for neutralization was highly toxic. so, before starting the experiment, we should aware of the chemicals what we use. Because they may be corrosive and toxic, so we should take some safety measurements before starting the experiment, like wear lab coat and safety glass and gloves. So that we can avoid some accidents and hazards. The chemical properties and physical properties of each chemical we use are explained as following: (reference: science direct)
TABLE NO. 1
-It is highly flammable.
-It is irritating due to defatting on skin.
-inhalation of acetone vapour may irritate respiratory system.
-Placing the container in well ventilated area.
-keeping the ignition sources away from it.
-if in case of eye contact, wash immediately with more water and seek medical advice.
Boiling point of it is very low and it is highly flammable
Shouldn't empty in to drains. Doesn't expose to source of ignition.
It is highly toxic. Inhalation of it or swallowing may affect the cardiovascular system.
Take the precautionary measurements against static discharge.
Sodium hydroxide (NaOH)
Contact with eye may cause long time sight loss.
In case of eye contact, wash eyes with more water and seek medical advice.
Sodium phosphate buffer
Its boiling point is very low and flammable.
Close the container with lid after using.
Sodium dodecyl sulphate
May cause skin allergy, highly flammable.
In case of skin contact Immediately flush skin with plenty of soap.
Multiple reaction monitor, an Agilent zorbax SB-C18 column (12.5 mile meters length, 0.2 mile meters of internal diameter, 5 micron meters of film thickness), fused silica capillary column, liquid chromatography/ mass spectrometry, gas chromatography/ mass spectrometry, 10ml test tubes, centrifuge test tubes, 10 micron grams spatula, one 1000ml (1 litre) cylindrical beaker, three 100ml beakers, glass rod, 5ml test tube, 1 litre cylindrical plastic bottle to store NaOH, thermometer, micro wave oven, centrifuge machine, Portable precision laboratory balance (reference: min shen, ping xiang).
1(a) SAMPLE PREPARATION FOR LIQUID CHROMETOGRAPHY/ MASS SPECTROMETRY:
The anabolic steroids hormones are extracted by using human hair, because anabolic steroids are more concentrated in hair than any other organ in human body and also easy to extract. Animal hair could be used for this experiment, because every living being contained anabolic steroids hormones. For sample preparation we use some amount of human hair, The hair sample is taken in to 10ml test tube up to half of it and then the hair is washed or rinsed with 0.1% of sodium dodecyl sulphate and make it dry using micro wave oven for 10 minutes (reference: ping xiang, min shen). Again the hair sample is washed with 1ml of water and 1ml of acetone, so that the hair will be free from shampoo, sweat, sebum, dust etc….,. The sample should be dried using micro oven by maintaining the temperature at 50 degrees Celsius for 10 minutes or till the water get evaporated. After the hair is get dried, cut the hair sample in to 1mm segment and weigh the 10mg of hair sample using Portable precision laboratory balance and take it in to a clean test tube. The hair sample were digested with 1ml 0f 1M NaOH about 10minutes at the temperature of 95 degree Celsius, for the heating operation we use hot oil bath by raising the temperature up to 95 degree Celsius. 1M NaOH is prepared by dissolving 40g of NaOH in distilled water and make up to 1liter gives 1M NaOH (reference: ping xiang, min shen). Sample will be cooled using cold tap water, it should be neutralized with 1ml of 1M HCl and 2ml of sodium phosphate buffer of ph=6.8. Sodium phosphate buffer is prepared by weighing 46.4g of sodium dibasic and 53.6g of sodium monobasic and both are dissolved in distilled water. Following Hcl and sodium phosphate buffer, 3.5ml of diethyl ether is added and the mixture is centrifuged at 2500rpm for 3minutes (reference: ping xiang, min shen). The organic layer from the sample should be transferred in to 5ml glass tube. The collected organic mixture is evaporated in the stream of nitrogen at 60 degree Celsius to get dryness. The obtained residue is reconstituted in 100 micron litres of acetonitrile and ammonium acetate buffer taking at the portions of 73:27 v/v. From this sample 5 micron litres were injected in to Lc/Ms. All sample prepared for the experiment are stored in freezer at 4 degree Celsius, so that no steroids will escape in to air (reference: ping xiang, min shen).
(b). LIQUID CHROMETOGRAPHY/MASS SPECTROMETRY SYSTEM SETUP:
For the liquid chromatography/mass spectrometry system contains an Agilent HPLC 1100 system with quaternary pump, an auto sampler and online degasser equipped with an MDS Sciex API 4000 triple quadrupled mass spectrometer. For instrument control and data acquisition, the analyst 1.4.1 software package is used. The column used in the lc/ms should be C18, 150mm of length , 2.1mm of internal diameter and 5 micron meters of pressure (reference: min shen, ping xiang). The C18 column is most common one, which was used for many experiments. The mass spectrometer should operated in the positive electro spray ionisation mode and the ion spray voltage should
maintained at 5KV and the temperature should be at 500 degree Celsius (reference: min shen, ping xiang). The only nitrogen gas is used as the curtain gas, the turbo gas, the nebulising gas. The declustering potential is optimized as the ion spray voltage, curtain gas and the nebulising gas all are used under default modes. For each analyst, multiple reaction monitoring is used for the multi product ion. The experiment's dwell time and mass width were set as 0.05 and 1amu (reference: min shen, ping xiang). The run time of the experiment is set as following, The initial time of the experiment is 3minutes up to 23minutes. The mobile phase gradient is used for resolution of the analysts. At run time 0 minutes , the methanol flow rate should be 73% , acetonitrile flow rate should be 0% and ammonium acetate buffer should be 27%. At 7minutes , methanol flow rate is 75% , acetonitile flow rate is 10% and ammonium acetate buffer flow rate is 15%. But at run times 8 minutes and 12 minutes the flow rates of all the compositions are same , flow rate of methanol is 50%, acetonitrile is 45% and the ammonium acetate is 5%. Again at run times 13 and 18 minutes the flow rate are same as for methanol 73%, for acetonitrille 0% and for ammonium acetate buffer it is 27% (reference: min shen, ping xiang). The system is run for 10 minutes at initial conditions. He flow rate of the column is maintained at 200 micron litres per minute. This experiment approximately takes 25 minutes. The compositions of LC mobile phase gradients are clearly showed in below table.
LC run time (minutes)
Ammonium acetate buffer (%)
(Reference: ping xiang, min shen)
2(a). SAMPLE PREPARATION FOR GAS CHROMETOGRAPHY/ MASS SPECTROMETRY:
Sample preparation for GC/MS is same like for LC/MS, but slightly different. Here also we wash the hair sample with 0.1ml SDS (sodium dodecyl sulphate) and dried. Again washed with water and acetone. After the sample drying, 50gm of hair sample is weighed using Portable precision laboratory balance and then taken in to a new clean test tube and it is digested with 1ml NaOH for 10 minutes using hot oil bath by raising the temperature up to 95 degree Celsius. Again sampled is cooled, this homogenate is neutralized by 1ml of 1M Hcl and 2ml of sodium phosphate buffer of ph=6.8 (reference: min shen, ping xiang). This solvent extraction is carried out with 3.5ml diethyl ether and then centrifuged at 2500 rpm for 3 minutes. Now we will get an organic layer, see that no solid particle is present in that. This supernatant is taken in to in a new test tube and evaporated using hot oil bath by raising the temperature up to 60 degree Celsius; it is heated for 30 minutes. after it is dried, it is derivatized by adding 50 micron litres of MSTFS (N-Methyl-N-trimethylsilyl trifluoro acetamide)/ iodotrimethyisilane/ DL - dithiothreitol, all of these are taken in portions of 1000:5:5,v/v/gm and heated in same hot oil bath about 80 minutes at 80 degree Celsius. all of these are taken in portions of 1000:5:5,v/v/mg. The final obtained sample is injected in to GC/MS system (reference: min shen, ping xiang).
(b). GAS CHROMETOGRAPHY/ MASS SPECTROMETRY SYSTEM SETUP:
Gas chromatography/ mass spectrometry analyses is setup with ion spray of 5 kilo volts and operate in the positive electro spray ionization mode (EI) on an Agilent 6899 gc and the source temperature is set at 500 degree Celsius. The Column should be fused silica capillary column with 0.25 mille meters of internal diameter, 30m and 0.1 micron meters of film thickness. The system operating conditions are helium inlet flow should be 1 milli litre per minute and sample injection should be split less and time for injection is 1 minute. The initial temperature for 2 minutes is set to 180 degree Celsius. The temperature will ramped up to 224 as 3 degree Celsius per minute and again the temperature will increased as 15 degree Celsius for every minute up to 300 degree Celsius. The sample injecting temperature is set at 250 degree Celsius. The source temperature is set at 220 degree Celsius and transfer line temperature is 300 degree Celsius. Stet the scan range from 3 to 450, because molecular weight of DHEA is 432 and testosterone is 435. Now the system ready to run the sample. This operation will takes approximately 25 minutes same like for Liquid chromatography/ mass spectrometry. The multiple reaction moniter will show the transition of each analyte and there collision energies (reference: ping xiang, min shen).
D. RESULT AND DISCUSSION:
1. EXPERIMENT PROCEDURE:
I have referred various literatures in order to identify an extraction protocol of steroids hormones from human hair. This review (using Science direct) resulted in several articles with such an extraction protocol. More than 20,000 articles are published only on steroids in hair. Since the experiments are done on hair of human being from various parts and also on animal hair like pig, horse. For this experiment i have chosen only the methods to extract the sample from hair from those various articles. The various articles reviewed for this research are mentioned in the table: 4. The method which i was felt a good one and its esay method to extract the sample and to identify the anabolic steroid in that sample published by MIN SHEN, PING XIANG, on 15th October 2009, which was the recent one among all. Some information about anabolic steroids was taken from other articles. Some methods mentioned in the table will take some days to get the results and some methods will take fewer hours to complete. I have followed the article which could done more faster and accurately. The extraction of anabolic steroids from pig hair was done by injecting or doping drug in to pig. The anabolic steroids under goes certain changes as time passes and these changes also can be identified by using liquid chromatography/ mass spectrometry and gas chromatography/ mass spectrometry. The anabolic steroids in the hair sample under goes a complex metabolism, which was depended mainly on the structural variation and application of pathways (reference: D. Thiemme, J. Grosse).
Analysis of anabolic steroids in human hair (LC/MS)
Newed deshmukh, iitaf hussain
Page no. 710 - 714.
Analysis of anabolic steroids in hair
Min shen, ping xiang
15th October 2009,
Page no. 773 - 778.th hkkkkgghghh
Testing of anabolic steroids in hair
Page no. S29 - S33
Physiological concentration of anabolic steroids in human hair
Min shen, ping xiang
30th January 2009.
Testing for anabolic steroids in hair from two body builders
Pascal kintz, H. Sachs.
13th may 1999,
Page no. 209 - 213.
Identification of anabolic steroids in seram, urine, sweat and hair.
D. thieme, j. Grosse
25th april 2003,
Page no. 299 - 306.
Analysis strategy for detecting doping agent in hair
D. thieme, J. Grosse
10th january 2000,
Page no. 335 - 345.
Doping control for nandrolone using hair analysis
Pascal kintz, cerenilli
March 2001, volumn 24.
Detection of physiological concentrations of cortisol and cortisone in human hair.
Jean - sebastien, pascal kintz.
The sample extraction contains various steps and operations like heating, cooling, drying, centrifuging, incubation, liquid extraction, derivatization etc....,. The sample extraction was done using human head hair. we can also used urine and other tissues for steroids detection. But if we use urine for the experiment, we can detect only recent drug abuse. And also not steroids are present in urine. But with hair we can identify steroids or drug abused long back. That is why we use hair samples for steroids detection. The hair in every part of our body has shaft and a root. Here the shaft contains of three principle parts. The inner medulla is made of poly hedral cells. The poly hedral cells consists of granules of eledin and air space. The central or middle cortex forms the amain part in the shaft among other two. The middle contex contains elongated cells, which consists of pigment granules in dark hair, but mostly air in white hair. The outer layer of shaft is called cuticle of hair. This consists of single layer of flate, thin and scale like cells which are most heaver and keratinized. The different component in the sample will have different absorption characteristics in a solid or liquid phase medium. The extraction of sample should do very carefully. For me, the preparation of sample took like 2 hours and I followed each and every step carefully. some operations takes more time to do. The stepwise procedure of sample extraction is explained as below.
I have taken some amount of hair sample in to a tube.
I washed this hair sample with 0.1% sodium dodlcyle sulphate.
After rinsing, i have dried the sample using micro wave oven at 50 degree Celsius for 10 minutes.
After it gets dried, i rinsed the sample with 5ml of distilled water and 5ml of acetone.
Again i have dried the sample.
After drying, the 10 mille grams of hair sample was taken in to a clean and clear 10ml test tube.
This taken hair sample was digested by adding 1ml of 1M NaoH about 10 minutes at the temperature of 95 degree Celsius. The heating operation was done on the hot oil bath by raising its temperature to 95 degree Celsius.
1M NaOH is prepared by adding 40gms of sodium hydroxide in water and maked up to litre.
After digesting, the sample was cooled by facing external surface of test tube to tap water for 3 minutes.
After it gets cool, sample was neutralized with 1ml of 1M Hcl and 2ml of sodium phosphate buffer.
The sodium phosphate buffer is prepared by dissolving 46.3gm of sodium dibasic and 53.7gm of sodium monobasic in to one litre of distilled water.
Following these, exactly 3.5ml of diethyl ether was added and well mixed through the bottom using glass rod.
This mixture was centrifuged at 2500rpm for 3 minutes.
Now at this step, the solid particles are get separated from the organic solution due to centrifugal force.
The organic layer obtained was shifted to a new test tube.
Then it was heated in presence of stream of nitrogen at 60 degree Celsius till it got dried.
After drying, some ml of diethyl ether was added and heated.
The residue was reconstituted in 100 micron litres of acetonitrile and ammonium acetate buffer.
From this, 5 micron litres were injected in to Liquid chromatography / mass spectrometry.
But for gas chromatography/ mass spectrometry, i did the derivatization, because of the keto and hydroxyl groups in their structure, so anabolic steroids will not show good chromatography separation behaviour that is why I did derivatization before going for gas chromatography / mass spectrometry.
The final sample was derivatized with 50 micron litres of mixture of N - methyl - N - trimethyl sliflouractonamide/ Iodotrimethyisilane/DL- dithiothreitol are taken i portion of 1000 micron litres/ 5 mille litres/ 5 mille grams.
After derivatization, it was injected in to Liquid chromatography/ mass spectrometry.
Liquid chromatography/ mass spectrometry will take approximately 25 minutes.
The mass spectrometry was maintained at positive ion spray
The voltage of ion spray was 5 kilo volts.
The temperature of source was maintained at 5oo degree Celsius.
The measurements of the column used for LC/ MS was 2.1 mille meter internal diameter, 150 mille meter length and 5 micron meters of width.
Initially the temperatures are maintained at room temperature.
After that, the initial temperature was maintained at 200 degrees Celsius for 2 minutes.
The temperature was gradually increased from 200 to 240 degree Celsius as 3 degree per minute
From 240 degree Celsius it again increased to 300 degree Celsius as 15 degrees per minute.
The source temperature was maintained at 200 degree Celsius.
The composition of LC / MS mobile phase gradient is adjusted as mentioned below. (reference: min shen, ping xiang).
TABLE NO. 3
LC run time
AMMONIUM ACETATE (%)
(Reference: min shen, ping xiang)
The different concentrations of steroids present in the extracted sample are depending on their physicochemical properties and also on their functional groups (reference: min shen, ping xiang). The each steroid present in the sample will have one hydroxyl group, which is very difficult to incorporate in to hair (reference: min shen, pig xiang). Due to presence of keto group and hydroxyl groups in the structures, the steroids don't show a better chromatographic separation behaviour (reference: min shen, ping xiang). To overcome this, i did derivatization before going to gas chromatography/ mass spectrometry. The derivatization done by using N - methyl - n - trimethyl slilflouroacetamide/ iodo trimethyisilane/ DL - dithiothreitol are taken in compositions of 1000 micron litres/ 5 mille litres/ 5 mille grams (reference: min shen, ping xiang). Not all steroids are detectable in Liquid chromatography/ mass spectrometry; some like boldenone, nandrolone, methandienone, trenbolone, methenolone, methyltestosterone are identified using LC/ MS. With the help of gas chromatography/ mass spectrometry we can identify remaining steroids like dehydro epiandrosterone and testosterone. The condions of gas chromatography/ mass spectrometry are maintained as below.
Gas chromatography was performed in the positive electron impact mode.
The time taken for injecting the sample was 1 minute.
The helium flow rate was 1 mille litre per minute.
Initial temperature was maintained at 180 degree Celsius for 2 minutes.
The temperature was set to increase gradually.
The temperature ramped to 224 degree Celsius at 3 degrees per minute.
Again the temperature increased to 300 degree Celsius at 15 degrees per minute.
Source temperature was maintained at 220 degree Celsius.
The nebulisation gas, curtain gas and ion spray voltage were kept at default mode and used.
Multiple reaction monitor was used to multiple product ions of each analytic.
The dwell time and mass width were 0.05s and 1 amu.
The measurement of the column used for gas chromatography/ mass spectrometry was 0.25 mille meters of internal diameter, 0.1 micron meters of film thickness and 30 minute.
Here the column I got for the experiment was 150 mille meters length, 2.1 mille meters of internal diameter and 5 micron meters of film thickness, which is most common column used for all experiments. Actually gas chromatography is most widely used technique used to separate atomic sized particles in a gas based on their molecular weight (reference: ping xiang, min shen). Before starting the experiment we ensure that no solid particle is present in the sample. First i start with liquid chromatography/ mass spectrometry, which is a reproducible, specific and sensitive method for the quantitative determination of eight anabolic steroids in human hair. With the used of LC/ MS we can detect steroids not only of human hair but also animal hair like pig, horse etc...,. Already so many experiments are done using animal hair. There is no much difference in both procedures and also in sample extraction. Liquid chromatography/ mass spectrometry took time approximately like 25 minutes. After liquid chromatography/ mass spectrometry i went for gas chromatography/ mass spectrometry, by derivartizing the sample.
EXTRACTION OF STEROIDS FROM THE SAMPLE
. LC-MS and GC-MS ANALYSIS OF STEROID EXTRACT:
I have done liquid chromatography/ mass spectrometry first, because derivartization process have to do for gas chrometograpy/ mass spectrometry with same sample, so i used some amount of sample for LC-MS and remaining for gas chromatography/ mass spectrography. For The specific and qualitative assay is essential to support analysis of the extract from hair sample. i have derivartized the sample why because, the anabolic steroid doesn't show the good chromatography because the presence of keto and hydroxyl groups ( reference: min shen, ping xiang), so the extract was derivatized before doing gas chromatography/ mass spectrometry. The extract consists of various chemical with different molecular weights, solubility, structure, boiling points. The composition of each chemical in preparation of extraction is very important and should be accurate. The quantity of sample Up to neutralization was 6 mille litres and up on heating it was came to 2 mille litre. Among all chemicals, diethyl ether has less boiling point that is 35 degree Celsius, so it was evaporated first and faster than remaining chemicals present in sample. The final quantity of the sample was come to 2 mille litres. Actually the final sample should be liquid in condition, but here the final sample here i got in semi solid. Here mass spectrometry will not allow the solid samples. This semi solid sample may contain the solid particle. After repeating the same experiment i have got the sample in liquid condition and then i have injected the sample split less in to liquid chromatography/ mass spectrometry of quantity 2 micron litres. The various collision and reaction of analytes are monitored on multiple reaction moniter. Each anabolic steroid hormone will have its own molecular weight and mass. Steroids are identified by their molecular weights and time of retention. But here, i was not been possible to detect anabolic steroids in liquid chromatography/ mass spectrometr. This may be because of mistakes took place while extracting sample from hair or difference in composition of chemicals used for it. After this, the anabolic steroids detection was done manually with respective their molecular weight and time of retention with the help of multiple reaction monitor. The reason for this might be mistakes took place while doing extraction of sample. In neutralization of sample we used diethyl ether, but we can also use pentane instead of diethyl ether. After repeating the same experiment procedure i got the sample in liquid condition. Eventhough i was unable to find the presence of anabolic steroid hormones by doing liquid chromatography/ mass spectrometry and gas chromatography/ mass spectrometry. Expect dehydro epiandrosterone and testosterone remaining steroids are detected in liquid chromatography/ mass spectrometry. Using gas chromatography/ mass spectrometry we will detect dehydro epiandrosterone and testosterone. But on gas chromatography/ mass spectrometry also steroids are not identified; then the experiment done manually of tracing the analytes based on their retention time. This may be because of the mistake took place while extracting the sample from hair. The peak of every steroid at their retention time are not obtained accurately. There are eight steroids with different molecular weights and retention time. The multiple reaction monitor of liquid chromatography/ mass spectrometry has shown the collisions of analytes and their retention time as following; the molecular weight of boldenone is 287.1, as per protocol its retention time was 5.47 minutes, but here we didn't see presence of boldenone steroid at retention time of 5.47, it means the sample has no boldenone in that. Similarly, the molecular weight of nandrolone is 275.2 and its retention time is 5.71 minutes, but the multiple reaction monitor didn't show nandrolone at that particular time. The molecular weight of trenbolone is 271.2 and its retention time is 4.98 minutes, but here also it was not found. The entire experiment was scanned manually, but no steroid was identified.
For gas chromatography/ mass spectrometry, the sample was derivartized before performing the experiment, because the sample contains keto and hydroxyl groups. The derivartizing agents are N - methyl-N - trimethyl silyltriflouro acetamide / iodotrimethyisilane and DL - dithiothreitol. Using gas chromatography/ mass spectrometry we can detect presence of dehydro epiandrosterone and testosterone. Using multiple reaction monitor we can see the collisions and reaction of each steroids and also can find their retention times. It is not been possible to find anabolic steroids in human hair sample. The problem might be, the derivative agents will volatile the sample, so that the steroids might escape in to free space, that was why no steroids found in both the liquid chromatography/ mass spectrometry and gas chromatography/ mass spectrometry. The molecular weight of dehydro epiandrosterone is 432 and its retention time is 15.73 minute, but no steroid was found at that time period. Similarly, the molecular weight of testosterone is 435 and its retention time is 17.11 minutes, same as before there was no steroids found. The molecular formulae of both the dehydro epiandrosterone and testosterone are same.
Steroids are unable to detect Using the gas chromatography/ mass spectrography , due to various problems occurred while doing the experiment to prepare sample like vary in concentrations chemicals, buffers prepared by me, chemicals exposed to room temperature etc...,. even the same thing has happend with liquid chromatography/ mass spectrometry. But in liquid chrometrography / mass spectrometry i have done derivartizing process before doing the experiment. I have used N - methyl-N - trimethyl silyltriflouro acetamide / iodotrimethyisilane and DL - dithiothreitol as derivartizing agents. The 50 micron litres of the sample is transferred in to 5ml test tube, the composition of each derivartizing agent is N - methyl - n - trimethyl slilflouroacetamide 1000 micron litres, iodo trimethyisilane milli litres, DL - dithiothreitol 5mille grams are taken in to a small tube and this composition was mixed with the 50 micron litres of the sample and heated up to 60 degree Celsius for 30min. For first 3 times the mixture was solidified due to unequal composition of the derivartizing agents. After using the derivartizing solution making up with correct composions of chemicals, the actual sample was formed and this final sample was used for liquid chromatography/ mass spectrometry. Even though the steroids are not found in the sample. This may be due to high volatile properties of the derivartizing agent. i came to know that, while heating the sample containing dervartizing solution in the final stage at 60 degree Celsius for 30mins the steroids are evaporated in to open space.
The liquid chromatography/ mass spectrometry and gas chromatography/ mass spectrometry both are sensitive methods developed and described as very suitable for detection and quantification of anabolic steroids in hair sample. These methods are useful in doping control. Actually these eight anabolic steroids (methyltestosterone, stanozolol, methandienone, nandrolone, trenbolone, boldenone and DHEA) could incorporate in to the hair and widely distributes in to hair shaft. The concentrations of these steroids are related to their physicochemical properties. These two methods are very useful in detecting the steroids doped by wrestlers or other sports men during the competitions and also could identify steroids abused long before using hair and urine samples. This experiment will provides the basic data on the time course and dose of the anabolic steroids depends up on steroid deposition in hair sample. The data obtained from this experiment will gives the ground work for further search. Actually there are so many steroid hormones present in human body and in other living beings like corisol, cortisone, methyltestosterone, methylone, etc..., but only some steroids are useful in body development.