An Ultra Fast and Sensitive Detection of 165 Drugs of Abuse in Human Urine Using Polarity Switching Ultra Performance Liquid Chromatog- Raphy Tandem Mass Spectrometry
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id15306796 pdfMachine by Broadgun Software - a great PDF writer! - a great PDF creator! - http://www.pdfmachine.com http://www.broadgun.com AAnnaallyyttiiccaaIlSlS N : 0974-7419 Volume 15 Issue 8 CCHHEEAMMn InIdIiSSanTT JoRuRrnYaYl Full Paper ACAIJ, 15(8) 2015 [319-338] An ultra fast and sensitive detection of 165 drugs of abuse in human urine using polarity switching ultra performance liquid chromatog- raphy tandem mass spectrometry Sachin Dubey, Shobha Ahi, Alka Beotra*, Tejinder Kaur, Shila Jain National Dope Testing Laboratory, Ministry of Youth Affairs and Sports, CGO Complex, Lodhi Road, New Delhi, 110003, (INDIA) E-mail : [email protected] ABSTRACT KEYWORDS The screening of wide variety of prohibited substances in a time bound Bioanalytical method; manner by adhering to latest World Anti-Doping Agency (WADA) guide- Sports drug testing; lines is a challenging task for doping control laboratories. The revised crite- UPLC-MS/MS; rion of detection limits (WADATD2013MRPL) has further required the doping Polarity switching; laboratories to review their testing procedures. The present work was aimed WADA. at developing a fast, sensitive and robust analytical method based on solid phase cleanup (SPE) and ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) to achieve the required detection lev- els. The method development involved optimization of deconjugation of phase II metabolites, SPE using mixed-mode ion cartridges for extraction of analytes of wider chemistries; and fast polarity switching UPLC-MS/MS detection. The developed method was validated to detect approximately 165 compounds and/or metabolites prohibited by WADA. The eight min- utes runtime allowed testing of approximately 180 samples in 24 hours at the limit of detection (LOD) of 50% below required detection levels. 2015 Trade Science Inc. - INDIA INTRODUCTION follow[2]. The prohibited list covers nine pharma- ceutical classes of substances (e.g., anabolic ste- The detection and identification of prohibited roids, corticosteroids, stimulants, diuretics, anti-es- compounds and methods of doping has been regu- trogens etc), three forbidden doping methods (e.g., lated for sports drug testing laboratories by World substance for enhancement of oxygen transfer, chemi- Anti-Doping Agency (WADA). The WADA publishes cal and physical manipulation and gene doping), and â-blockers are a prohibited list every year consisting of wide range two groups of analytes alcohol and of pharmacological classes of drugs[1]. WADA cre- prohibited in specific activities[1]. Therefore, numer- ates respective technical documents that outline mini- ous technical approaches are needed to analyse the mum required performance limits (MRPL) as well great diversity of doping agents. as international standard for laboratories (ISL), Anti-doping analysis is conducted in two steps. which accredited doping control laboratoriesmust Initially, screening of samples is performed, in the 320 An ultra fast and sensitive detection o.f 165 drugs of abuse in human urine ACAIJ, 15(8) 2015 Full Paper case of a suspiciousresult; an additional selective pre-treatment for a screening purpose is a challenge confirmation is carried out[3]. As every sample has as the method should provide good extraction yields to be screened, the screening method has to be highly for a combination of analytes with very different sensitive and specific to ensure identification of sus- physico-chemical properties (neutral, basic and pected sample and in the same time should mini- acidic, lipophilic and hydrophilic). Moreover, hu- mize the probability of false suspects. Doping analy- man urine is usually a very dirty matrix so it re- sis requires the use of several different chromato- quires an extraction technique in which the impuri- graphic, mass spectrometric and immunological ties of the sample should be eliminated to avoid any methods[4-7] which makes it mandatory for all the possible unwanted interference of the matrix. The doping control laboratories to have a number of sepa- diversified categories of drugs of abuse mostly con- rate analytical procedures, thereby making screen- tain basic compounds with exceptions of neutrals ing of each sample more complex, time-consuming (glucocorticosteroids) and acidics (diuretics). Be- and laborious. Therefore, it has become necessary sides, the huge number of different endogenous com- to develop high-throughput techniques to screen in a ponents normally found in urine makes the selective single method a large set of compounds with differ- detection of analytes at low concentration very chal- ent physicochemical properties avoiding false nega- lenging. Recently, various methods have been de- tives and false positive results. veloped to detect the banned compounds in human In the last decade, the suitability of liquid chro- urine using dilute and shoot approach[18-19]. The di- matography-tandem mass spectrometry (LC-MS/MS) lute and inject approach is certainly fast, but it does has been demonstrated as a technique of choice over not allow for the detection of analytes at very low traditional gas chromatography mass spectrometry concentrations and does not include deconjugation (GC-MS) methods for multi-target screening due to of glucuronides[20-21]. Hydrolysis is mandatory to re- the development of electrospray ionization (ESI) move the glucuronide moieties attached to several – sources which operates at atmospheric pressure[8- doping agents during phase II metabolism. Liquid 11]. This capability allows the detection of both low- liquid extraction (LLE) may be used for the sample and high-molecular weight compounds[12]. purification but it faces some pitfalls. To cover a The ultra performance liquid chromatography wide range of different drugs, two consecutive ex- (UPLC) based methods for screening analyses have tractions, one at basic and the other at acidic pH, is emerged in the anti-doping field. The advantages of required. In addition, LLE requires careful separa- µ UPLC (in which columns are packed with sub-2 tion of the phases and can be time consuming and particles operating at pressures up to 1000 bar) have tend to use large volumes of solvent. Moreover, the been demonstrated to rapidly and efficiently sepa- sample extracts are not very clean. rate drugs and related substances[13,14]. Due to the The most preferred technique for said require- reduced analysis time, peaks become very narrow ment is solid-phase extraction (SPE) as it requires and an adapted detection device is thus mandatory. less washing and cleaning steps and blocks the im- Mass spectrometers with fast scanning like triple- purities of the sample within the cartridge. A single quadrupole (QQQ) are often coupled to UPLC and or multiple-stage SPE has been applied for the used in tandem mode by monitoring ion transi- sample preparation methods in various fields of dop- tions[15,16]. Modern QQQ instruments offer very fast ing[20-22]. In particular, a single mixed-mode cartridge acquisition cycle times and polarity switching usu- is reliable for the fractionation of acid, neutral and ally expanding the number of analytes which can be basic drugs from biological samples because the detected in a single run. Numerous methods have drugs are adsorbed separately by hydrophobic or been employed in the past decade conjoining UPLC ion-exchange interaction on to the cartridge. with QQQ mass spectrometer (UPLC-MS/MS)for To ensure consistency of the measurements identification of variety of doping agents[15-17]. amongst doping control laboratories, WADA defines The choice of development of a suitable sample the MRPL, which is the concentration of a prohib- Anallyttiicall CHEAMn InIdSianT JoRurnYal ACAIJ, 15(8) 2015 Alka Beotra et al. 321 Full Paper ited substance at which the laboratories are expected fast analytical method based on multi target approach to detect the prohibited drug/s. From January 2013, reducing the burden of number of detection methods WADA has revised the MRPL criterion for the de- and analysis time 2) method sensitive enough to tection of drugs by the anti-doping laboratories achieve the detection levels. Hence, the aim of this wherein, the MRPL of various drugs has been re- work was to develop a high throughput and sensi- duced from 20-80% (TABLE 1)[23]. In addition, the tive screening method for the detection of various criteria for limit of detection (LOD) is also revised drugs at or below the WADA TD2013 MRPL. It was ’s (TDMRPL2013) which states that the laboratory required to derive the extraction procedure to ex- method validation of the initial testing procedure tract analytes with a very wide range of chemistries shall include the estimation of the LOD for each com- as well as the detection method so as to improve the pound and the estimated LOD shall not be higher LOD of drugs. than 50% of the MRPL. The revised MRPL and LOD Hence, a three step strategy was made for the criterion applicable from January 2013 has further overall improvement of the method: i) Enzymatic necessitated the need to review and revise testing hydrolysis for the deconjugation of phase II metabo- procedures in the doping laboratories to achieve the lites (glucuronides), ii) SPE using mixed mode car- targeted MRPLs. tridges for the execration of acidic, basic and neu- The lower detection limits enforced in the new tral molecules in single step and, iii) identification technical guidelines of WADA has required improv- of drugs on highly sensitive and upgraded instru- ing the existing screening method