Liquid chromatography (LC) and mass spectrometry (MS) were developed as an alternative way of testing other than GC/MS which was used to do tests of nonvolatile liquids. GC/MS is gas chromatography/mass spectrometry (Tsuchihashi 2005). LC/MS is used in forensic testing and clinical toxicology.
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When correct phases of either mobile or stationary are used correctly, there can be good results of LC separations of polar analytes. They include amines, carboxylic acids, etc. LC is used mostly because it analyzes liquid samples (Garg 2010). Drugs are mostly abused in sports. Performance-enhancing drugs are the most abused drugs (Bonini & Plebani 2009). Urine is liked by many for testing drugs because it is collected in large volumes.
The drugs can be detected even after three days in urine unlike in blood.
The presence of synthetic steroids was a major setback in the testing of drugs until LC/MS was introduced. Drugs like glucocorticosteroids are tested using LC/MS method. There are others like stimulants and anabolic steroids. These drugs are not mostly detected by other methods like GC/MS (Hoofnagle 2010). The paper has two different examples of how LC-MS is used in drug screening to understand better.
Instruments Used for Screening Drugs
The instruments are Varian ProStar 410 AutoSampler. The second one is Varian ProStar 210 Isocratic Solvent Delivery Modules and the last one is Varian 1200L LC/MS which has an ESI source (Jemal 2004).
Materials and reagents
Standard solutions; are supposed to be 1.0 mg/ml of 1S, 2R Ephedrine, Amphetamine, and Methamphetamine. Standard solutions include Amphetamine-D5, MDEA-D5, and MDA-D5 (Steffenrud 2009). Vacuum pumps and racks are also needed (Larry 2006). A rack is used for holding test tubes.
Standard solutions of aliquot which measured 100 μL each were transferred to labeled tubes. Then urine was added, 1 ml with 0.1 N KOH which measured 0.1 ml. The mixture was then added well with a vortex (Thevis 2005).
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The mixture was then transferred to a Sorbent bed which was activated with a 3 ml Focus cartridge. It was before mixed with methanol which was 1ml (Robert 2005). The bed was then flooded with deionized water which was 1 ml. This was passed under a vacuum. There is further washing of the bed with 2× 1 ml acetonitrile under a vacuum (McMaster 2005).
An autosampler vial was used to collect the resulting solution with 2 × 100 μL elution solvent. The elution solvent included acetonitrile and formic acid.
The bed which was Sorbent was washed with water measuring 600 μL (Wilfried 2006). It was flooded under a vacuum to remove any elution solvent. This water was also used to dilute the solution for injection (Yergey & Alfred 2006). The solution was later injected with an aliquot which was 10 μL for analysis.
The column is monochrome MS 5 μm, 50×2mm. The mixer is a 250 μl static mixture (Thurman 2004).
Solvent A is a 0.2 % formic acid. Solvent B is methanol. Injection Volume is 10 μL and the injection Solvent can either be methanol or formic acid (Henion 2005).
The Ionization mode was ESI positive (Yo 2005). The collision Gas was 2.0 m Torr Argon. API Drying Gas was 30 psi at 380 degrees Celsius (Yo 2005). The API Nebulizing gas was at 59 psi. The scan time was 1.8 Sec and SIM Width was 0.7 amu. The needle was 5000V and the shield was 600V. The capillary was 30V and the detector was 1800V (Brotherton 2005).
Combined Results of LC/MS Study of Amphetamine Drugs
|Drug Name||min||RSD |
From: Thermospray liquid chromatography-mass spectrometry of corticosteroids’, J Chromatography, vol. 48 no. 5, pp. 52-59.
Discussion and Conclusion
The LC testing took 6 minutes to complete a full cycle. At 1.65 minutes, the test had shown its first peak (Brotherton 2005). At 4.12 minutes, there was completion of the remaining peak. Amphetamine drugs produce two ions which were used for analysis while MSA and MDMA produced multiple ions (Brotherton 2005). The final eluent was finally injected into an LC/MS system for screening. More than 85% of the drugs were recovered from the urine (Brotherton 2005).
Materials and Methods
There were reference standards and Steraloids. Reference standards came from Sigma, USA and Steraloids came from Newport, USA. There was Acetonitrile which came from Mumbai, India. Methanol came from Phillipsburg, USA. Ethyl acetate came also from Mumbai, India (Brotherton 2005). Butyl methyl ether came from New Jersey, USA and formic was purchased from Mumbai, India (Brotherton 2005). There was De-ionized water and organic solvents the reagents were in good condition (Brotherton 2005).
LC and MSMS Apparatus
The apparatus contained an Agilent 1100 series (Yo 2005. There was also a pumping system which pumped at high pressure (G1311A). There was an Autosampler (G1329A) and G1316A column compartment. It was also important to add degasser (G1379A). Diode array Detector (G1315B) was also used (Yo 2005). Triple Quadruple instrument (API 3200) was used to do mass spectrometric (MS). It was put to work with a heated nebulizer; a source of ionization (Yo 2005). The below table summarizes the analytical parameters that were used in the MS. The analyst 1.4 software was applied to monitor the process (Yo 2005).
|Mass spectrometer||working parameters|
|HPLC working parameters|
|Column||C-18 ODS Inertsil, 50 × 4.6 × 3.5 μm|
|Flow||700 μl per minute|
|Injection volume||10 μl|
|Mobile phase||1% Formic acid, Acetonitrile|
|Gradient||0-5 min B 15%, 5-6 min B 60%, 6-7 min B 100%, 7-7.10 min B 15%|
From: The evolution of mass spectrometry in the clinical laboratory’, Methods Mol Biol, vol. 60 no. 3, pp. 1–7.
Sample Preparation Procedure
There was a procedure used which was to isolate drugs based on their acidity, alkalinity, and neutrality. Isolation was done on a urine sample before the process of LC/MS occurred (Neels 2006).
In the MS, the mobile phase was put through electrospray (Neels 2006). Nitrogen acted as both the curtain and nebulizing gas. It led to fragmentation. Each dwell time took 60 msec (Neels 2006). The LC temperatures were at 25 degree Celsius.
Ten microliters with the testing solution were added to urine that was free of drugs. This was to prepare the calibration standards to achieve the concentration levels that were required (Neels 2006). Glucocorticosteroids that measured 30ng/ml and stimulants amounting to100ng/ml consisted of the control samples (Neels 2006). There was 17α-Methyl testosterone which was prepared to act as an internal standard (Neels 2006). Working solutions were obtained when methanol was diluted with methanol.
There was room for accuracy because the test had 12 control samples.
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The method detected 28 drug agents. There was a good recovery percentage, 57-114. The drug compounds were like diuretics and steroids which were seen when the test exhibited different conditions of P.H (Oreans 2006). This was seen when there was a comparison of means of processed samples which were ten with those of unprocessed reference solutions (Neels 2006). All of them had equal concentrations.
The LC-MS was capable of detecting 28 different drugs. The method separated in a short duration and produced good results (Hoofnagle 2010). LC-MS detected a lot of drugs including volatile and non-volatile liquids, unlike the other methods which limited their testing to specific drugs (Neels 2006). The other methods like GC-MS only detected drugs like diuretics, specific anabolic steroids, etc while LC-MS did testing to more than 14 glucocorticosteroids and more anabolic steroids (Neels 2006). It detects other drugs like stimulants, some estrogenic drugs, etc hence detecting like 28 drugs types.
Diuretics include chlorothiazide and steroids include 3-OH-stanozolol. Examples of stimulants include methylphenidate and mesocarb (Neels 2006). Each drug exhibits different ionization patterns, the same for the fragmentation of drugs.
After doing the LC-MS testing, glucocorticosteroids were found to be neutral compounds when ionizing.
Steroids were polar and fragmented in a short time (Neels 2006). This is the same for diuretics. Stimulants were seen to be sensitive. Formic acid was used in the mobile phase same with acetonitrile.
Challenges and Solutions of using LC/MS system
The challenges of the LC/MS system range from the complexity of the tests done to detect specificity problems, the high cost of maintaining instruments, insufficient detection, and a lot of work. Sponsors also find it expensive to use the LC/MS tests and sometimes they find their time consuming (Ramsteiner 2006). The tests require experienced personnel hence a new person is required to spend 3-6 months training on how to use the instruments (Hoofnagle 2010). These challenges have been a source of hindrances for effective testing.
In recent years, instruments have been introduced which are less expensive, handle a lot of work, work efficiently, and do not require a lot of attention to maintain (Maurer 2008). Some tests are done at affordable prices (Hoofnagle 2010). In conclusion, they are fewer tests that are done to minimize the time taken. Instruments have been manufactured to multitask to take less time.
Bonini, P & Plebani, M 2009, ‘Errors in laboratory medicine’, Clin Chem, vol. 48 no. 3, pp. 691–700.
Brotherton, H 2005, ‘Determination of drugs in blood serum by mass spectrometry/mass spectrometry’, Anal Chem, vol. 55 no. 4, pp. 549-553.
Garg, U 2010, ‘The evolution of mass spectrometry in the clinical laboratory’, Methods Mol Biol, vol. 60 no. 3, pp. 1–7.
Henion, J 2005, ‘High-speed liquid chromatography/tandem mass spectrometry for the determination of drugs in biological samples’, Anal Chem, vol. 58 no. 5, pp. 2453-2460.
Hoofnagle, A 2010, ‘Peptide lost and found: internal standards and the mass spectrometric quantification of peptides,’ Clin Chem, vol. 56 no. 8, pp. 15–25.
Jemal, M 2004, ‘High-throughput quantitative bioanalysis by LC/MS/MS’, Biomed Chromatogr, vol. 14 no. 3, pp. 22–25.
Larry, D 2006, ‘Analytical advances in detection of performance enhancing compounds’ Clin Chem, vol. 43 no. 7, pp. 299–304.
Maurer, H 2008, ‘Liquid chromatography-mass spectrometry in forensic and clinical toxicology’, J Chromatogr, vol. 7 no. 4, pp. 13–25.
McMaster, C 2005, LC/MS: a practical user’s guide, John Wiley, New York.
Neels, H 2006, ‘Evaluation of the drug profiling system’, Clin Biochem, vol. 32 no. 3, pp. 409-412.
Oreans, M 2006, ‘Column switching in high-pressure liquid chromatography’, J Chromatogr, vol. 83 no. 5, pp. 267–277.
Ramsteiner, K 2006, ‘Systematic approach to column switching’, J Chromatogr, vol. 5 no. 2, pp. 45-47.
Robert, D 2005, Liquid chromatography-mass spectrometry: an introduction, Wiley, London.
Steffenrud, S 2009, ‘Thermospray liquid chromatography-mass spectrometry of corticosteroids’, J Chromatography, vol. 48 no. 5, pp. 52-59.
Thevis, M 2005, ‘Mass spectrometry in doping control analyses, Curr Organ Chem, vol. 98 no. 2, pp. 45–48.
Thurman, E 2004, Liquid chromatography/mass spectrometry, MS/MS and time of flight MS: analysis of emerging contaminants, American Chemical Society, Columbus.
Tsuchihashi, H 2005, ‘Determination of drugs in human urine by liquid chromatography-mass spectrometry’, J Anal Toxicol, vol. 20 no. 2, pp. 281-286.
Wilfried, M 2006, Liquid Chromatography-Mass Spectrometry, CRC, Boca Raton.
Yergey, A & Alfred, L 2006, Liquid chromatography/mass spectrometry: techniques and applications, Plenum Press, New York.
Yo, M 2005, Analytical methods in forensic chemistry, Ellis Horwood, Chichester.