Chromatography News (A Publication of Chromatographic Society of India)(A Publication of Chromatographic Society of India)

Analytica Anacon 2011, Special IssueAnalytica Anacon 2011, Special IssuePresident's Message

Chromatographic Society of India is very pleased to bring out this special issue of “Chromatography News” to coincide with the Analytica Anacon Trade show being held in Mumbai from October 12-14, 2011.

There are two important topics that have been appearing in the press very frequently, one being the topic of Doping in Sports and the other being the destruction caused by the terrorism. However, these press reports do not get into the details of the substances used as the doping agents nor the chemicals used as explosives in the terrorism. The importance of Chromatography and Mass Spectrometry lies in the analysis and identification of these chemical substances used both in doping and terrorism. While it is easy in the case of sports doping to find out the details of the chemical agents used by any individual; in the case of terrorism it is

possible only to find out the chemicals used in the explosion that can perhaps lead to its origin and the people behind it. It can be simply said that chromatography and mass spectrometry are of immense help in bringing the culprits in both the above areas.

CSI is very pleased in contacting the right scientists, best suited to write authoritatively on these topics; they are Dr. Shila Jain, Director of the National Dope Testing Laboratory (NDTL), New Delhi to give us the right perspective on doping, the social responsibility and the importance of Chromatographic and mass spectrometric techniques in the identification of the doping substances. Similarly, Dr. Rukmani Krishnamurthy, who has retired as the Director of Forensic Science Laboratory, Mumbai is an expert in the identification of the explosives used in the terrorism and how the chromatographic and mass spectrometric techniques help in bringing the culprits into the hands of law. Therefore, this issue of 'Chromatography News” is very important to raise the awareness of our readers and go beyond the news media to understand more about these two topics of national interest and importance.

This issue also contains important information of the new HPLC training course that CSI will be conducting in Collaboration with the Dr.P.S.Ramanathan Advanced Instrumentation Centre housed in Ruia College, Matunga on October 17th and 18th, 2011. This training will be imparted by the expert faculties of CSI and this should benefit both the beginners of HPLC and also those using this technique in the Pharmaceutical Industry. Similarly, CSI is organizing a one day seminar on the important topic of Bio Analytical Research and Application. The varied contemporary topics of the Bio Analytical field will be dealt by the experts, who are also senior managers in reputed CRO laboratories in the country. During this seminar scheduled for 8th November, 2011 in MTNL Training Centre Auditorium in Powai, Mumbai,CSI is organizing a Lifetime Contribution Award to Professor (Dr) R.T.Sane, who has been a Pioneer in the area of Bio Analytical Research and the one who has guided more than 150 students for M.Sc and Ph.D. degrees. Many of his students, today are well placed in the pharmaceutical, chemical industries , academic and research institutions.

CSI hopes that by engaging the experts from the academia and industry on the various Chromatography techniques, it will be possible to conduct frequently seminars and workshops in various parts of the country. It is also hoping to build the renewed website to be a source for education in chromatography. There will be concise slides on the theory of various chromatographic techniques and their instrumentation and applications. The website also will contain the list of chromatography products, their manufacturers and the subsidiaries in the country and their Distributors. CSI is also planning to upload the slides on the selection criteria of chromatographs and mass spectrometers and also some of the tender specifications that are usually published, for the ready reference of the prospective buyers. It is also the intent of CSI that it should be able to guide the users of Chromatographic instruments to help with information that they need wherever possible. Further, the website is also linked to the UK publication, 'Chromatography Today’, that has many application articles and reviews that are of immense use to any chromatographer, this is the direct benefit of the collaboration the CSI has established with the Chromatographic Society of UK. We urge all readers to visit our website www.chromscienceindia.org for more details.

Lastly, CSI is a registered non-profit organization, which is committed to education in chromatography and mass spectrometry and solicits individual chemists to become its Life members and companies selling instruments, consumables and reagents for Separation Science and companies using chromatography heavily and believing in the Corporate Responsibility to come forward and become Corporate Members thus supporting this cause for chromatography education.

Dr.G.RamakrishnanPresident, CSI

Importance of Chromatography and Mass Spectrometry in Doping Analysis in Sports

In pursuit of gold and glory, large numbers of sportspersons all over the world are using performance-enhancing substances in an effort to gain competitive edge over the others. Sports are about fair play and Fair play demonstrates attitudes and behaviours in sports consistent with the belief that sports is an ethical pursuit. It does not include acts of violence, cheating, drug abuse or any other form of exploitation in an effort to win. When elements such as these are introduced, the true competitive spirit is lost, and there remains no purpose for sports. Doping is the deliberate or inadvertent use by sportspersons of a substance or method banned by Medical Commission of International Olympic Committee/WADA.

The Role of Gas Chromatography and Mass Spectrometry in Doping Analysis.Anti-doping analysis is a very important and interesting area of forensic toxicology, aimed at detecting the abuse of prohibited substances and methods by the athletes. Doping control analyses using instrumental analytical tools have been performed since the 1960s, when various measures were initiated to control the misuse of performance-enhancing drugs in sport. The analysis of stimulants began after gas chromatographic separation was introduced. These substances could be effectively detected by chromatographic methods, especially by gas chromatography (GC), a technique which, at that time, was rapidly developing as one of the most powerful in analytical chemistry

Mass spectrometry has played a decisive role in doping analysis and doping control in human sport for almost 40 years. With its unique sensitivity and selectivity properties, mass spectrometry provides state-of-the-art technology in analytical chemistry. The Olympic Games which play an important role in International sport today are milestones for technical development in doping analysis. The mass spectrometric techniques that are most commonly used are Gas chromatography mass spectrometry (GCMS) using typical bench top Quadrupole system (GCMS) and for lower detection levels GC/MS/MS with ion traps or GC/HRMS (High resolution mass spectrometry) using magnetic sector instruments. In recent years Liquid chromatography (LC) coupled with mass spectrometry (LC/MS) has also been used more often for detection of corticosteroids, diuretics, beta-blockers, anabolic steroids, heat labile stimulants, narcotics etc. A very specialized form of mass spectrometry called Isotope Ratio Mass Spectrometry (IRMS) is now being used by many WADA laboratories to detect doping with endogenous steroids such as testosterone. GC coupled to IRMS (GC/IRMS) could therefore be used to discriminate between the endogenous and synthetic origin of naturally produced steroids, mainly T and/or its precursors and metabolites, since the synthetic compounds have less 13C than their endogenous homologues

In general, MS is now applied in WADA-accredited laboratories, following separation by either LC or GC, with ionization techniques such as Electron Ionization (EI), Chemical Ionization (CI), Electrospray ionization (ESI), Atmospheric Pressure Chemical ionization (APCI) and Atmospheric Pressure Photoionization (APPI). The mass spectrometer configuration ranges from low resolution Quadrupole and Ion-Trap techniques to high resolution Magnetic Sector, Time-Of-Flight (TOF) and FT instruments including Orbitrap techniques. Multiple Stage Mass Spectrometry (MSn) experiments are achieved either in Triple-Stage Quadrupole (TSQ) , Q-Trap, or Ion-Trap (IT) instruments, including Linear Ion-Trap and Orbitrap instruments with collision-induced dissociation (CID), electron transfer dissociation (ETD) and Pulsed Q dissociation (PQD). Both target analysis and general approaches for unknown doping agents are applied

Dr. Shila JainDirector, NDTL

Equipment Utility in Doping

GC/MS To detect anabolic steroids, beta agonist and few stimulants, narcotics, cannabinoids

GC/MS/MS or GC-HRMS To detect low concentration of anabolic steroid

GC/NPD/MS To detect Stimulants and narcotics

LC/MS/MS To detect corticosteroids, diuretics, beta blockers, stimulants, narcotics and other heat labile drugs , peptide hormones

GC-IRMS To differentiate between natural or synthetic origin of endogenous steroids

The advances in chromatography and mass spectrometric techniques have brought the offenders under the scanner and strict measures have been taken to disqualify those taking performance enhancing drugs and punish them by not allowing them to take part in the competition till they are cleared by the authorities. This has helped in ensuring a fair competition among the sports persons and also ensuring better health conditions of the athletes. .

Anti-doping programIn recognizing the achievements made towards effective doping control, it is of utmost importance to applaud the joint endeavors of the World Anti-Doping Agency (WADA), the International Olympic Committee (IOC), the international federations and national anti-doping agencies to combat doping. Advances against the misuse of prohibited substances and methods, which are performance-enhancing, dangerous to health and violate the spirit of sport, can be achieved only if all the stakeholders work together.

WADA based in Montreal, Canada, was established in 1999 as an independent agency composed and funded equally by sport movement agencies and governments of the participating countries in the world. Its key activities include scientific research, education, development of anti-doping programs, maintaining the WADA anti-doping code –the document harmonizing anti-doping policies in all sports and countries. WADA works towards a vision of a world where all the athletes compete in a doping free sporting environment.The Govt. of India with its commitment towards “Drug Free Sport” launched a scheme of Dope Test from the year 2002 for educating our youth and sportspersons about harmful effects of performance enhancing substances and also for testing of athletes by most advanced scientific methods.

As per the mandatory requirement of WADA, the autonomy of test planning and testing facilities needs to be maintained separately. With this aim, Indian Government has approved National Anti-Doping Agency (NADA) & National Dope Test Laboratory (NDTL) as separate autonomous bodies under Ministry of Youth Affairs and Sports. (MYAS). NADA has been given the role of Anti-doping program by implementing effective testing, education and result management in India; whereas, NDTL has been given the responsibilities of analyzing of samples as International Standards setup by WADA.

National Dope Testing Laboratory got the prestigious accreditation by World Anti Doping Agency (WADA) in Sept 2008. NDTL is one of the 33 WADA accredited laboratories in the world and 6th in Asia. The 2010 has been watershed year for NDTL in the terms of achieving higher credibility and acceptability at both national and international level by testing samples for Singapore youth Olympic Games-2010, Commonwealth Games-2010 and Asian Beach

Dr. Shila Jain is Scientific Director, National Dope Testing Laboratory (NDTL), Government of India, accredited by World Anti-Doping Agency (WADA) and Member, Medical Commission, Indian Olympic Association. She obtained her Ph.D. degree from P.G.I. Chandigarh and obtained advanced training in Doping analysis from Moscow, Singapore, Beijing, Cologne, London, Tokyo and United States. She has authored more than 50 research publications and attended conferences and Seminars at national and International level over the last 20 years. She has conducted Workshops, Courses for Educating players, Physical Educationists, Physiotherapists, Doctors and Administrators about the Drug abuse and Doping methods in Sports.

Games all within a span of 4 months. The website of NDTL, India (www.ndtlindia.com) is launched in Jan 2010. The testing protocol being done at NDTL has brought the lab amongst top ten labs in the world and will strive to continue to achieve the targets and goal set by the WADA in creating a drug free environment in India'

Commonwealth Games testing TEAM at NDTL

National Dope Testing Laboratory, New Delhi

Chromatography Techniques used to counter Hi-Tech Terrorist Blast cases.

Latest Technologies easily available to mankind have encouraged the Terrorist activities leading to Hi-Tech crimes in India. These are increasing day by day with sophistication in modus operandi. Mumbai the economic centre of India was attacked by a series of bomb blasts at twelve different places within a span of one hour on 12th March 1993. The main explosive used was RDX. After about 10 years, terrorist activity of late has again erupted taking a toll on innocent lives, with the use of explosives causing death and destruction. On 2.12.2010, a public bus at Ghatkopar, was blown up by an impoverished explosive device (IED) with two causalities. On 27.01.2003 the public vegetable market at Vile Parle was targeted causing heavy damage and panic among common people. On 13.03.2003 a fully packed local train compartment at Mulund railway station was blown up by an improved explosive device and the causalities ranged up to 10. In most of

the explosions, the explosives used were RDX, NC-NG, etc. The blast that occurred at the Zaveri Bazaar and the gateway of India on 25.08.2003 showed presence of big craters at the blast site and on the analysis the presence of RDX and petroleum oil were found. On 11th July, 2006, a series of seven bombs went off in trains, killing 209, which also showed the presence of RDX, ammonium nitrate and petroleum oil.. On 26th November 2008 to 29th November 2008 - Coordinated series of attacks, killing at least 172 people took place. On 13th February 2010 - a bomb explosion at the German Bakery in Pune killed fourteen people, and injured at least 60 more.people. On 13th July 2011 bombings took place at three locations in Mumbai.

Introduction:Forensic laboratories play an important role in the analysis of bomb Blast cases for the identification of bulk explosives from a defused device or explosive charge and residues at the trace level on the debris from craters, affected articles like damaged vehicle parts, broken glass and damaged clothes of the victims and on the penetrated missiles recovered from the bodies of the victim. In cases of bomb blast, exhibits are meticulously examined for post exclusive residues using the chemical spot test, HPTLC, HPLC,GC-MS and GC-Ion Scan. In cases of unexploded explosives infra-red spectroscopy is used as it is a finger print technique for explosive analysis. Post explosive analysis of traces left in the debris is a challenge to forensic scientists. In cases of severe blasts the most commonly used explosives are RDX, TNT, PETN, Tetryl, etc. In cases of small explosions the explosives used are generally potassium chlorate, gun powder and firework compositions. Chromatography Techniques are of great use in analysis of post explosive articles as it separates the different explosives and identification can by MS, IR etc

Method of Analysis1. HPTLC Analysis: The concentrated Acetone extract was spotted on pre coated HPTLC plates along with standard TNT, Tetryl, NCNG, RDX and PETN. The HPTLC plates were developed in three different solvent systems to get accurate results. Solvent systems used were 1. Toluene 2.Chloroform :acetone(9:1) and 3. Trichloroethylene:acetone (8:2). The developed HPTLC plates from solvent systems 1 and 3 were sprayed with alcoholic sodium hydroxide solution, then heated in an oven at 100 degree Celsius for 15 minutes, after cooling they are subsequently sprayed with Gries re-agent. A pink colored spot of RDX was visualized for all exhibits the HPTLC plate developed in solvent systems too was spread with thymol blue in concentrated sulphuric acid. Violet colored spots of RDX were visualized for all exhibits. Acetone extracts of all exhibits were evaporated to dryness under the current of the air. The residues were re dissolved in methanol and passed through a 0.45 um filter and filtered extracts were used for further instrumental analysis.

2.HPLC . Thermo separation product model coupled with a UV spectrophotometric detector, UV 6000 LP and an auto sampler. The software used was Chromquest.

Column: Lichrospher RP-C18Mobil Phase: Methanol:water (50:50)Flow: 1.5 mm/minWavelength: 230nmInjection volume: 10ul

The HPLC technique was also used for quantitation of a diffused charge of RDX, seized a few days after bomb blasts.

3. GC-MSAcetone extracts of all the exhibits were further separated and purify by preparative TLC using silica Gel –G(25um) as a stationary phase and chloroform:acetone(8:2) as a mobile phase. The separated band of RDX was scrapped and eluted with acetone. The eluted acetone extract was evaporated to 0.1ml and subjected to GC-MS. About 0.2 l of solution was injected.GC-MS model-Trace GC with Trace MS, Thermo Finnigan, UK.

Dr. (Mrs) R. Krishnamurthy Ex. Director, FSL, Mumbai

The MS Analysis was carried out in the E.I mode conditions:Column: Capillary HP-5, 15 m x 0.32 film thicknessOven Temperature: 100-200 degree CelsiusRamp rate: 10 degree Celsius per minFinal Temperature: 250 degree Celsius. Final hold -20 min.

4. GC-Ion Scan

Acetone extracts of all the exhibits were purified by preparative TLC and use for GC -Ion Scan Analysis GC Ions scan model, Barringer instruments, was used. In the GC-Ion scan the sample of explosive after separation by GC using a capillary column was introduced into the reaction by a carrier of dry air. The reaction region has a weak radioactive ionizing source Ni 63, B emitters and the Positive and negative Ions formed by collision of B particles in the sample stream. Explosives are electronegative substances and form negative ions. An ion mobility spectrometry (IMS) detector allows the negative ions to form by the explosive substance into the drift region. The various ions travel at different characteristic speeds and arrive at the collector electrode at a unique drift time. RDX has a drift time of 2.1 min. the sensitivity of GC-Ion Scan for RDX is 500 pg.

Results and Discussion.

GCMS of RDX

GC-Ion scan of RDX

LCMS of RDX

In conclusion, the analysis of explosive residues on vehicle parts and clothes is based on physical observation and chemical examination. In the case of organic explosive like RDX, crime scene evidences like crator formation, blackening and oil stains are indicative. The directions of bending of vehicle parts, damage of other parked vehicles, distance of missiles thrown from the blast site, condition of the CNG cylinder evidences reveal clues about the type of explosive used for blasting. The study of crators helps in deciding the nature of explosives. The observation of crime scenes indicates the explosive impact that is extent of damage was up to 300ft surrounding area. In twin explosions, hired motor taxies were used for carrying IEDS. The collection of the taxies, the dickey portions and CNG cylinders was damaged severely, separated and thrown at a very long distance from the blast site. The condition of of the dickey and formation of craters revealed that the bomb was kept in the dickey. The CNG cylinder added the severity of the effects in the explosion. The presence of blackening and oil stains on the damaged vehicles and metal pieces revealed that the culprits had mixed explosive in petroleum oil. No missiles were found at the crime scene. The use of heavy petroleum oil in the explosive charges posed problems in sample preparation and analysis. The extraction of residual explosive from petroleum oil was done by successive extracts with hexane which helped in getting rid of heavy petroleum oil. The interference of this oil in TLC, HPCL and GC-MS was very prominent.

Dr. (Mrs) Rukmani Krishnamurthy was the Director, Directorate of Forensic Science Laboratories,Home Dept,Govt. of Maharashtra. Mumbai for several years. She is presently the Technical Adviser to The Institute of Forensic Science, Higher & Technical Education Dept. Maharashtra State, She have started Diploma Courses in Forensic Science & Related Laws and Digital and Cyber Forensic & Related Laws and Three years B.Sc. in Forensic Science in Mumbai from the academic 2009-2010.Board of studies for Forensic Science have been approved by the Academic Council of Mumbai University. As the ex-Director at HELIK ( Global Education Research Institute) Mumbai, she has ventured into new concept of public- private partnership in establishing a Global Education Research Institute catering Forensic and allied services to Corporate sectors, IT Industry, Banks, Financial sectors, Insurance, Police, Judicial, etc.

Chromatography News (A Publication of Chromatographic Society of India)(A Publication of Chromatographic Society of India)

Chromatographic Society of India is pleased

to invite interested chemists

and students to enrol for the following courses

a) Two-day workshop on “HPLC as an Analytical Tool”organized by CSI in collaboration with Dr. P.S. RamanathanAdvanced Instrumentation Centre (PSRAIC) on October 17thand 18th, 2011 at Ruia College, Matunga, Mumbai

b) One-Day seminar on “Recent Trends, Challenges and Solutionsin Bio Analysis” by eminent speakers from the leading CROs inIndia on Tuesday, November 8th, 2011 at MTNL ManagementTraining Institute, Powai, Mumbai

For more details, please visit CSI booth C9c at the Analytica Anacon Trade Show or contact Ms. Renita Aposta at the following address:

Chromatographic Society of India219 Mastermind-1, Aaarey Milk Colony Goregaon (East), Mumbai - 400 065Phone/Fax : +91 22 28797522 E-mail : info@chromsocindia.org

(CSI is a non-profit organization committed to Education in Chromatography and Mass Spectrometry)

Chromatography News is a publication of Chromatographic Society of India. This issue is edited and published by Dr. G. Ramakrishnan, President of CSI. He may be contacted via email : info@chromsocindia.org