The simultaneous detection of narcotic analgesics and non-steroidal anti-inflammatory drugs in human urine using high performance liquid chromatography - tandem mass spectrometry
The use of over-the-counter products (OTCs) has increased over the years. This is evident from the wide range of products available and also the easy availability of these OTCs in pharmacies, health shops and even supermarkets in South Africa and elsewhere. Therefore the procurement of these products is easy and they are used by many consumers for self-treatment of numerous conditions. This has given rise to the problem of irresponsible use of these products by consumers. Pain is the most common pharmacological challenge encountered by the medical practitioner and therefore treatment for pain is frequently prescribed. Narcotic analgesics and non-steroidal antiinflammatory drugs (NSAIDs) are drugs used to relieve pain. The use and misuse of drugs and medications by competitors in sport has been recognized as an important problem. Ethical aspects of competing under non-equal opportunities are of concern. Elite athletes who turn to doping take the greatest risk to satisfy their burning desire for gold regardless of the health risks involved. Deaths under the influence of drugs and combinations thereof are not uncommon in sport. Athletes might use substances to eliminate any obstacle they might encounter during their training. One of the obstacles they might encounter is pain felt from injury or excessive training. The misuse of drugs for sport enhancement is a health risk because of the kinds of drugs used and the large doses given. The narcotic analgesics and the NSAIDs are some of the drugs abused by competitors to overcome the effects of pain. The aim of this study was to develop a screening method for the simultaneous detection of an extended list of narcotic analgesics and NSAIDs in human urine using high performance liquid chromatography – tandem mass spectrometry. An extended list of some of the narcotic analgesics and NSAIDs available in South Africa was compiled. Literature research was done on these reference substances and their metabolites. It was found that information from the literature was limited. The mass spectrometry properties of each reference standard were determined using a LCMS/ MS system in positive electron ionization mode. A new method was successfully optimised using these mass spectrometric properties. Information regarding extraction procedures for these compounds was collected. The extraction procedure included a hydrolysis step with β-glucuronidase/arylsulfatase followed by liquid-liquid extraction with diethyl ether at pH 7. No derivatization steps were necessary during sample preparation, which shortens the preparation time. This extraction procedure was successfully combined with the LC-MS/MS method for the screening of the list of narcotic analgesics and NSAIDs. The final chromatographic conditions for this study are as follows: Autosampler properties: Agilent 1100 Autosampler Injection volume: 10 μl Runtime: 20 minutes Pump method properties: Agilent 1100 Binary Pump Mobile phase: Solvent A: 0.01% formic acid Solvent B: acetonitrile Step Time (min) Flow rate (μl/min) A (%) B (%) 0 0.00 200 90.0 10.0 1 2.00 200 90.0 10.0 2 8.00 200 10.0 90.0 3 9.00 200 90.0 10.0 4 20.0 200 90.0 10.0 The final extraction procedure is as follows: · 2 ml urine · 1 ml acetate buffer pH = 5.2 · 25 μl ß-glucuronidase/arylsulfatase enzyme · Hydrolyse at 50 °C for 2 hours · Add 1 ml phosphate buffer pH = 7 · Add 5 ml diethyl ether · Shake horizontally for 5 minutes on a mechanical shaker · Centrifuge for 5 minutes · Transfer organic phase to an ampoule containing 50 ml internal standard (Apomorphine) · Evaporate to dryness · Dissolve in 100 μl mobile phase. Good results were obtained during the validation. Good specificity was obtained for each compound without the interference of the background or co-extracted compounds. Good repeatability was obtained for the listed compounds. The calculated CV% for all the compounds was under 15% for low, medium and high concentrations. The calculated LOD values was relatively low for most of the compounds, with exception of paracetamol, which has a high back-ground and piroxicam which could not be extracted effectively from the urine. The freeze and thaw stability of the listed compounds was satisfactory with only salicylic acid which was outside the range of 20% after 3 cycles of freeze and thaw. For long term stability the results were different. The results for the analysis of the control sample after 3 months of storage at -20oC were below 80% for 18 compounds when compared to the sample analysed immediately before storage. From these, 15 compounds were between 70 and 80% and 3 compounds were between 60 and 70%. The method was applied to obtain the excretion profiles for diclofenac, its metabolite, 4’- hydroxydiclofenac, and indomethacin. For diclofenac the peak excretion was determined at 6 hours after administration with a sharp decrease of excretion until 24 hours after administration. For its metabolite, 4’-hydroxydiclofenac, the peak excretion was also determined at 6 hours after administration, but with a slower decrease from 6 hours and can still be detected after 48 hours. For indomethacin the peak excretion was determined at 6 hours after administration with a slow decrease from 6 hours and can still be detected after 48 hours. A further application of this screening method was done by analyzing a series of 137 real urine samples collected from competitors from different sporting events. The results showed that there is a large number of these substances used by competitors in sport. The number of positive samples was 37% of the total number of samples tested. Rugby with 39% of the positive samples was identified as the sporting event with the highest usage of these substances, with paracetamol identified as the substance that is the most frequently used. It can be concluded that this method is effective in detection of the listed narcotic analgesics and NSAIDs in urine from human.